Flow cytometer

ABSTRACT

A Faraday cage enclosing the flow chamber of a cytometer and ground planes associated with each field deflection plate in concert therewith inhibit electric fields from varying the charge on designated events/droplets and further concentrates and increases forces applied to a charged event passing therethrough for accurate focus thereof while concomitantly inhibiting a potential shock hazard.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG-48 between the United States Department ofEnergy and the University of California for the operation of LawrenceLivermore National Laboratory.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention lies in the art of biomedical, scientific instrumentation.More specifically the invention relates to cytometers and instrumentsfor high speed identification and sorting of cells, organelles andchromosomes. In particular the invention discloses an improved cytometerevent deflection and sorting apparatus and method.

2. Description of the Prior Art

Various techniques of flow cytometry have been employed over the lastquarter century from an initial effort to count particulate matter in afluid environment, subsequently to size particles and more recently toquickly quantify multiple chemical, physical or structural properties ofcells and cellular composites of inhomogeneous populations. The firstsuch effort related to counting individual red cells in a liquidsuspension forced through a capillary glass tube on a microscope stage.Problems encountered by such means involved standardizing capillarytubes, assuring proper focus, maintaining even flow and obtainingappropriately sensitive photoelectric apparatus to accomplish anaccurate count.

Some of these problems were resolved by injecting the particlesuspension into a laminar sheath flow of fluid, which flow surroundedand aligned the particles, and thereby virtually eliminated largeparticle blockage and coated the particle stream. Particle count by suchmeans was accomplished by detecting the variation of electricalcharacteristic of the path through the laminar flow caused by theinclusion or exclusion of cellular matter therein. In addition particlesizing could be accomplished because pulse amplitude width was relatedto particle volume, and could be evaluated by pulse-height analyzers ornuclear pulse amplifiers. Photoelectric counting was later introduced.Subsequent cytometric application utilized spectrophotometry to quantifycellular constituents or alternatively to clarify cellular constituentsvia multiple simultaneous measurements of different cellular features,through UV absorption and photon scattering.

All the foregoing systems required a suspension of cells to pass througha constricted channel traversed by a beam of light orthogonal to saidchannel in which light intensity varied dependent upon position of thecell in the channel. Another possible variation, however, directed thelight beam parallel to the flow and made calculations based on lightscatter. Florescence at variable wavelengths or absorptioncharacteristics were later used to characterize DNA and RNA constituentsin the flow orthogonal to the illuminating beam.

Later cytometric improvements involved pneumatic, hydraulic andelectrostatic techniques to separate cells from a flow after photometricor electrical sensing. A following fluid switch cell sorter diverted astream by means of a sonic transducer that converted laminar flow toturbulent flow.

More recent efforts utilize a sheath fluid flow chamber to which iscentrally added a fluid flow of sample body cells or organelles inaqueous suspension. The flow chamber is vibrated at high frequency by apiezoelectric transducer which causes a sheath stream jet exiting theflow chamber with samples to break into discrete droplets from an exitpoint of the flow chamber. Upon exiting the flow chamber the jet anddiscrete droplets pass through electrical charging means that chargeeach droplet either positively or negatively as determined by laseridentification of samples or events in the sheath flow prior to dropletformation. The charged droplets then pass through a pair of verticalplates, one charged at a negative voltage and the other at a positivevoltage. The positively charged droplets shift stream toward thenegative plate and the negative droplets shift stream toward thepositive plate. Uncharged droplets continue in a straight line out ofthe flow chamber to a collector tube below.

Although the foregoing electronic charging of droplets allows forsorting of particles with two attributes by positive or negativecharging, there remains long standing need for identification of morephysical or chemical characteristics than presently exist in the art,and therefor more accurate sorting of events than is permissible withstate of the art deflecting mechanisms. As described above traditionaldeflection plates are rectangular shaped conductive plates of oppositecharge at ±3000 volts. The plates are ungrounded, uncovered andunshielded, thereby subjecting an operator to possible unwarranted andhazardous electrical shock. In addition, being unshielded, the strongelectric field of the deflection plates may affect charging of thecharged flow stream of event droplets at the point of inducing thecharge which can affect the charge and distort accurate deflection ofthe stream. Furthermore, the virtually straight lines of electric forcefrom positive plate to negative plate and perpendicular to the chargedstream flow does not allow for accurate stream deflection. Chargeddroplets can easily and without hindrance flow along parallelequipotential lines between the charged plates.

If the electric field were not only perpendicular to the stream flow,but also doubly curved back on itself by each oppositely charged fieldplate, the electric field would possess a plurality of force vectorsgenerally flowing in opposed directions, creating a focusing effect ofthe charged droplet stream. By putting a ground plate on each oppositelycharged deflection plate, in accordance with the invention an increasedand oppositely curved electric field with multidirectional force vectorsis obtained yielding a much more potent focusing force field than ispossible with the prior art unidirection electric field.

OBJECTS OF THE INVENTION

It is therefore a primary object of the invention to allow more accuratefocusing of a deflected droplet/event stream between field plates of acytometer.

Another object of the invention is to curve the electric field betweenthe field plates in order that force vectors from the field operating ona charged particle therein will be substantially increased and morevariable in direction and therefore more efficient and accurate insetting a desired direction of deflection and focus of the charged eventflow.

Another object of the invention is to provide for ground planesassociated with each charged field plate in order to curve the electricfields of each charged field plate back on itself and thereby confinethe field and effect a greater concentration and increased strength ofthe force field.

Yet another object of the invention is to diminish the potential ofshock hazard from either charged plate by providing proper ground andinsulation of exposed high voltage components.

Still another object is to provide a Faraday barrier between the strongelectric field of the field plates and the charged event flow stream aseach event is being charged upon exiting the flow chamber.

Additional objects, advantages, and other useful and novel features ofthe invention will become more readily apparent to one skilled in theart upon inspection of the attached drawing as clearly delineated by thefollowing detailed description of the invention and in light of theappended claims.

SUMMARY OF THE INVENTION

The invention is an improved method and apparatus for deflecting andsorting charged events or droplets of a fluid stream or aqueous flow ofbody cells and components thereof.

The invention apparatus utilizes conventional cytometer instrumentationconsisting of a flow chamber for creating a sheath fluid flow, a samplefeed for said sheath flow of an aqueous suspension of cells andorganelles to be sorted. The flow chamber is operated upon by apiezoelectric transducer to break up the fluid into a stream ofdroplets. At least one laser is focused upon the fluid flow to determinedesired events by means of florescent dye detection and scattered laserlight. As each desired event is detected, it is identified via computermemory bank of stored data, then tagged and segregated in each dropletwith a positive or negative charge and passed through a path ofrelatively parallel and oppositely charged deflection plates. Only oneevent per droplet is accepted; droplets with no events or multipleevents are not tagged or charged.

The invention deals specifically with the charged field plates utilizedfor deflecting/sorting appropriately charged events. Ground plane platesare combined with conventional oppositely charged deflection plates,with an insulating layer between each charged plate and its respectiveground plane. In addition each charged field plate face is wrapped witha nonconductive layer such as mylar tape to further prevent hazardousshock to an operator.

By such means electric fields are caused not only to flow in relativelystraight lines from positive to negative plates but also to followcurved trajectories in opposite directions from positive to ground andfrom ground to negative, the combination of which creates asubstantially increased and focused electric field.

A Faraday cage/shield is provided by a metallic box around the flowchamber and in particular between the flow chamber and highly chargedfield plates to prevent field interference with and affect on eachevent/droplet as it is being charged.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of prior art state of the art cytometerapparatus, showing in particular a cross section of existingfield/deflection plates.

FIG. 2 is a perspective view of the invention illustrating the Faradaycage and deflection plates with ground planes, and showing in particulara cross section of the new and novel field plates.

FIG. 3 is a cross section of prior art field plates illustratinguncertain focusing of charged events.

FIG. 4 is a cross section of the invention deflection platesillustrating accurate focusing of charged events.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Referring now to the drawings, FIG. 1 illustrates the cytometerenvironment to which the invention is applied. In FIG. 1, a flow chamber10 of the cytometer apparatus is fed by a sheath fluid tube 12 atapproximately 40 psi. An aqueous flow of cell samples, organelles orcomposites thereof is fed into flow chamber 10 under a similar pressureby a centrally situated sample tube 14. A sheath flow with samplescentrally aligned therein exits flow chamber 10 at a chamber nozzle andorifice 16 and forms a jet 18 of fluid approximately 0.5 mm long and 50μm wide. The flow chamber is operated upon by a piezoelectric crystaltransducer 20 which vibrates flow chamber 10, nozzle 16 and fluid jet 18at approximately 100 KHz, which causes jet 18 to undulate and break intoa stream or flow of droplets 22.

At least one laser 24 emitting laser beam 25, typically two or morelasers operating at different frequencies, are focused through optics 26on fluid jet 18. A second laser 21 emitting beam 23 is shown in FIG. 2.Typically samples have been coded with various dyes which have specificelectron excitation quantum levels and respective luminescence atspecific frequencies. Therefore, cells, chromosomes or other organellescoded by respective dyes can be excited or identified as desired eventsto be sorted out for further evaluation.

The luminescence 27 of the samples typically passes through focusingoptics in the manner of a lens 28 and pin hole 30, possibly through oneor more dichroic mirrors 32 if more than one luminescence color isindicated, and a standard mirror 34, through band filters 36 and 38 tofluorescence detectors 40 and 42 for different colors, e.g. red andgreen.

Typically at least one laser having light scatter 43 going to a detector44 is implemented to obtain additional characteristics, physical more sothan chemical, of the sample event.

When a desired event is observed, for example a particular chromosome ofa human cell, various of the foregoing detectors will so indicate, atwhich time an event/droplet 22 at the moment of breaking away from fluidjet 18 will be given a positive or negative charge, q+ or q-, byrespective electronics 46. Detectors relay information to a computerwhich utilizes a look up table to identify the event. With two or morelasers X and Y coordinates can be set up to adequately identify theevent in computer memory and as displayed on a CRT. The respectiveelectronics for identification and charging events is more clearly setout in U.S. Pat. No. 5,150,313 to Ger van den Engh et al., issued Sep.22, 1992.

Event/droplet stream 22 typically passes through a pair of oppositelycharged metal field/deflection plates 48 and 50 charged at a negative3000 volts and positive 3000 volts, respectively. An electric field (E)49 is thereby created from positive plate 50 to negative plate 48,perpendicular to the charged event flow 22. If an event/droplet ischarged positively in fluid jet 18, positive event stream 51 will bedeflected by the electrostatic force F=(q+)E, and caused to flow into afirst sample tube 52 and correspondingly a negatively charged eventstream 53, will be deflected by the electric field force F=(q-)E andcaused to flow into a second sample tube 54. Noncharged droplets arecollected in a third tube 56.

As can be observed from FIG. 1, event/droplet formation and charge onfluid jet 18 can be affected or acted upon by stray electric field 49between field plates 48 and 50. In addition, deflected droplet/eventstreams 51 and 53 can only be deflected to either positive or negativefield plates 50 and 48, respectively, but cannot be confined to adesired and focused plane. These handicaps or limitations tend to beeliminated by the invention disclosed herein.

A perspective view of the cytometer improvement invention is illustratedin FIG. 2. FIG. 2 illustrates only the flow chamber and deflection plateportion of FIG. 1. In FIG. 2, flow chamber 10, is protected fromspurious electric fields by a Faraday cage/shield 58 which consists of ametallic box having three sides 60, 62 and 64 and a floor 66 and beingopen at the front and top for operator and equipment access andobservation. A first, hole 68, is provided in side 64, for a first laserbeam 25 from first laser 24 to be focused approximately 150 μm downstream on fluid jet 18 exiting orifice 16 of flow chamber 10. A secondlaser 21 focuses a second laser beam 23 approximately 150 μm down streamfrom first beam 25.

Detection of forward laser scatter 43 by scatter detector 44 is madepossible by a second hole 70 in side 60, and detection of laserluminescence 27 of events in fluid jet 18 are made possible through athird hole 72 in side 62.

A fourth hole 74 is provided in floor 66 of Faraday shield 58 forpassage therethrough of event/droplet stream 22. As will be observed,spurious electric fields are prevented from entering shield 58 andthereby are prevented from affecting the charging of events/droplets 22upon breaking away from jet 18.

Event/droplet stream 22 is caused to pass through first and secondfield/deflection plates 48 and 50, which are charged to a -3000 voltsand a+3000 volts, respectively. The inner surfaces 76 and 78 of fieldplates 48 and 50 are set on either side of event flow 22 at a slightlydiverging angle of 2°-3°. First and second metallic ground planes 80 and82 coupled to a common ground are wrapped around the outer surfaces andsides of field plates 48 and 50, and are separated therefrom by aplastic or other nonconductive spacer 84 and 86. Provision for groundplanes 80 and 82 effects the substantial change in the electric fieldbetween the charged plates 48 and 50 enabling very accurate focusing ofcharged event flows 51 and 53, as will be further illustrated andexplained in FIGS. 3 and 4.

FIG. 3 is a cross section of prior art field plates 48 and 50 of FIG. 1.FIG. 4 is a cross section of the invention field plates 48 and 50 ofFIG. 2; i.e. the field plates of FIGS. 3 and 4 themselves may beidentical rectangular sheets of metal, but the added ground plane of theinvention in FIG. 4 effects novel and more advantageous features thanexists in earlier deflection field plates.

In FIG. 3, which is the view that the charged event/droplet stream 22sees as it passes between field plates 48 and 50, droplet 22 observesparallel lines of force (E) going from positive plate 50 to negativeplate 48 perpendicular to parallel equipotential lines (EP) both ofwhich are orthogonal to the direction of travel of droplets 22. Ifdroplet 22 is positively charged (q+), lines of force (E) will forcedroplet 22 to the left toward negative plate 48 to, for example,equipotential line a; however, once on line a or any other equipotentialline for that matter, nothing can stop positive droplet 22 from driftingup and down said equipotential line as indicated by dashed droplets online a. It cannot be determined with any degree of accuracy exactlywhere positive droplet 22 will focus and fall.

Correspondingly, if droplet 22 is negative (q-), lines of force (E)would force the droplet to the right toward positive plate 50. Againsaid droplet, however, is free to shift and travel along anyequipotential line such as c, and is so indicated in FIG. 3 by dasheddroplets.

Referring now to FIG. 4, where a cross section of the invention fieldplates is illustrated, a much more complex electric field is observed asdroplets 22 travel down the track between field plates 48 and 50.Droplet 22 again observes parallel line of forces from positive tonegative plates 48 and 50 and corresponding parallel equipotential linesx,y, and z; however, droplet 22 also observes curved lines of force onpositive plate 50 traveling from all points on plate 50 to ground plane82 on either side thereof, and further observes curved lines of force onnegative plate 48 going from ground plane 80 on either side thereof toall points on negative plate 48. In effect, another equipotential lineαβ is created along the line of deflection of which droplet 22 is causedto travel. Due to the unique curvature of ground plane lines of force,droplets can be deflected and can only be deflected along EP line αβ.

With the addition of ground planes 80 and 82 to field plates 48 and 50,substantially more potential energy is capacitively contained within thesystem, concentrating and effecting more lines of force and thereforegreater force in deflecting droplets that pass therethrough. Theinvention system operates not only as an event sorter but also asfocusing lens for charged particles.

Between the field plates of FIG. 4, a negatively charged event/dropletcan only travel along line αβ to positive plate 50; correspondingly, apositive charged event/droplet can only travel along αβ toward negativeplate 48. There is no possibility of charged particle drift on anyequipotential line and with increased and concentrated lines of force,the deflection is accomplished in a substantially more efficient manner.

The foregoing description of preferred embodiments of the invention havebeen presented for purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teaching. The embodiments were chose and describedin order to best explain the principles of the invention and itspractical application to thereby enable others skilled in the art tomake and best utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of the invention be defined by the claimsappended hereto.

I claim:
 1. Cytometer apparatus for sorting cells and particles,comprising:a. a flow chamber providing a flow of charged particles, b. aFaraday shield enclosing said flow chamber, c. a pair of oppositelycharged deflection plates disposed on either side of said chargedparticle flow, each having an internal surface facing said particleflow, an opposed external surface, and vertical sides, d. ground planessurrounding and spaced apart from the external surface and verticalsides of each said deflection plate, and e. insulation means located inthe space between each of said charged deflection plates and itsassociated ground plane.
 2. Cytometer apparatus according to claim 1,wherein said Faraday shield is provided with passages therethrough foroperation upon said charged particle flow by at least one laser. 3.Cytometer apparatus according to claim 2, wherein said Faraday shield isprovided with means for detecting laser scatter and event luminescence.4. Cytometer apparatus according to claim 1, wherein said deflectionplates comprise a pair of rectangular shaped conductor plates orientedlongitudinally along said particle flow.
 5. Cytometer apparatusaccording to claim 1, wherein each said internal surface of saiddeflection plates is covered with a nonconductor.
 6. Cytometer apparatusaccording to claim 1 wherein the flow chamber includes means for formingfluid droplets, and the charged particles are contained in saiddroplets.
 7. Cytometer apparatus according to claim 1 wherein thedeflection plates are charged to about +3000 V and -3000 V respectively.